Technical Field
The present disclosure relates to a method for analyzing lipoproteins. Particularly, the present disclosure relates to a technique capable of calculating the concentration of cholesterol ester or free cholesterol, and further, the concentration of lipoprotein particles with high reliability from the actual measurement data obtained by analyzing lipoproteins.
Description of the Related Art
The following Patent Literature 1 describes a method for classifying lipoproteins contained in a subject sample according to the particle size by means of gel filtration liquid chromatography and then for quantifying cholesterol and triglyceride (triacylglycerol) being contained in the classified lipoproteins. According to this method, the lipoproteins are fractionated into chylomicrons, very low density lipoproteins, low density lipoproteins, and high density lipoproteins by subjecting the obtained chromatogram to waveform processing such as a Gaussian distribution curve approximation.
The following Patent Literature 2 discloses a method for classifying lipoproteins contained in a subject sample according to the particle size by means of gel filtration liquid chromatography and then for quantifying cholesterol and triglyceride being contained in the classified lipoproteins, wherein the lipoproteins are classified into 20 subclasses.
However, the classifying method disclosed in the Patent Literature 2 has been applied only to a case where a specific column is used, and respective peak positions for the 20 subclasses (i.e., particle size) have not been supported with theoretical foundation. For example, a particle size of LDL (low density lipoproteins) is determined to be 25.5 nm as a cutoff value when a GGE method (polyacrylamide density gradient gel electrophoresis) is used, and is determined to be about 20 nm based on a size observed by an electron microscope when a NMR method is used, and is determined to be 25.5 nm based on a size obtained by a GGE method when a gel filtration FPLC method is used, and is also determined to be 25.5 nm based on a latex bead or spherical proteins when a gel filtration HPLC is used. The particle size may vary depending on ways of estimation when a light scattering method is used. In addition, this particle size may become a different value by determining a molecular weight in accordance with equilibrium ultracentrifugation.
Therefore, definition of the lipoprotein subclasses based on the particle size and comparison of respective lipoproteins contained in a serum sample between these subclasses will lead to confusion.
In view of the foregoing circumstances, the present inventor made the following proposals in the following Patent Literature 3:                Use, as a reference, a peak position observed in a metabolic disorder patient whose lipoprotein metabolism suggests that the size ranges of VLDL, LDL, and HDL are narrow.        The use of the aforementioned reference peak position makes it possible to perform a comparison between the subclasses of a plurality of samples even if different gel filtration columns having different specifications of separation and molecular size range are used.        
The above proposals made it possible to determine the component peaks of 20 subclasses independently of the type of a column.